A micro TAS technique of forming a micro flow channel in a substrate, flowing specimen DNA and a reagent into it to give rise to a biochemical reaction has been and being used in various technical fields and the usefulness and the advantages thereof are well known.
A technique of using an intercalator type fluorescence label for detecting the quantity of double-stranded DNA in a micro flow channel is also known. With this technique, double-stranded DNA is heated to about 50° C. to 90° C. and the temperature at which the double-stranded DNA dissociates into single-stranded DNA is determined to know the type of DNA by seeing the change of the fluorescence intensity.
However, a micro flow channel is very small and a cross section thereof shows a side thereof that is as short as several μm to hundreds of several μm. When observing the fluorescence intensity in a micro flow channel, while varying the temperature of the flow channel, the substrate is warped due to temperature changes and the flow channel is displaced. The substrate is deformed and the flow channel is displaced to a large extent particularly when the substrate is made of a plastic material. Additionally, the influence of auto-fluorescence of the substrate material should preferably be avoided when detecting weak fluorescence from a fluorescence label in a flow channel. For this purpose, preferably, the imaging area of the imaging device is minimized so as to pick up only fluorescence from a fluorescence label without receiving any auto-fluorescence. This can be achieved by means of a technique of raising the NA of the imaging optical system to reduce the depth of focus, a technique of detecting light on the basis of the principle of confocal imaging or that of light sectioning to reduce the imaging depth or some other similar technique. However, when the imaging depth is reduced by means of these techniques, fluorescence cannot be detected accurately unless the flow channel is so aligned that the imaging depth is accurately located in the flow channel. Additionally, there can arise a problem that fluorescence cannot be observed accurately because of a shift of the flow channel plane partially from the scanning plane due to deformation of the substrate, a manufacturing error of the flow channel, a positioning error and so on.
It may be conceivable to provide the imaging device with an auto-focusing mechanism as a measure for solving these problems. Japanese Patent Application Laid-open No. 2001-242081 discloses an auto-focusing mechanism that operates on a real time basis. The device is mounted on a device for detecting the fluorescence label bound to the probe of a DNA chip so as to receive excitation light reflected from the surface of the DNA chip by means of a quadrant photodiode and it is determined if the focal position is in front of or behind the target by seeing the differences of the quantities of light received by the four elements of the quadrant photodiode. Then, the lens position is adjusted to constantly keep the chip surface in focus.
Japanese Patent No. 3551860 discloses a device having an imaging system formed by using one-dimensional sensors (multi-channel photomultiplier tubes) and provided with an AF (auto-focusing) mechanism. Japanese Patent Application Laid-open No. 2006-322707 discloses a method of detecting the upper and lower walls of a flow channel by means of a quadrant photodiode and scanning between them in a zigzag manner.
The device disclosed in Japanese Patent Application Laid-open No. 2001-242081 is a device of a double scanning system. More specifically, a spot of excitation light is formed on a target of detection and produced fluorescence is led into a light amount detection element such as a photomultiplier tube (PMT) so as to acquire information on the entire surface of the target of imaging, while shifting the relative position of the spot of light on the target of imaging. This system is disadvantageous when the target of imaging is driven two-dimensionally for scanning because the scanning operation is time consuming and the device is bulky. Additionally, it is unrealistic to move a flow channel substrate containing liquid in the inside because bubbles may appear and liquid may leak out. The above-cited patent document also discloses a method of driving an optical head that includes an objective lens to be used for projecting excitation light and converging fluorescence, relative to a fixed target of imaging. While this method can drive the optical head to scan more quickly by means of a small drive unit if compared with moving a target of imaging, the use of a drive unit is a prerequisite for it so that the device is inevitably large and it is not free from the problem that the imaging operation is time consuming. Furthermore, it requires the use of a sensor dedicated to AF (auto-focusing). Still additionally, the optical head to be operated for scanning is inevitably heavy and the scanning operation is time consuming because the voice coil motor for driving the objective lens in the direction of the optical axis needs to be driven with the objective lens at the time of scanning operation.
The device disclosed in Japanese Patent No. 3551860 has an imaging system and a focus detection optical system and is adapted to only adjust the focus relative to a single spot. The focus is fixed during a scanning operation. Therefore, the focus cannot be adjusted on a real time basis to accommodate the positional shift of the flow channel while scanning a line.
The scanning method disclosed in Japanese Patent Application Laid-open No. 2006-322707 is a method to be applied to a double scanning scanner. It is not efficient because the scanning operation using it is time consuming.